Abstract Aim The phylogeographic relationships among populations of the common Cape River crab, Potamonautes perlatus, are examined to investigate whether the contemporary population genetic structure is congruent with the hypothesized hydrographic evolution of drainage systems established during the Pliocene, or whether it reflects an older Miocene climatic amelioration. Location 139 samples of P. perlatus were collected from 31 populations distributed among the five major perennial drainage systems and a number of smaller catchments in the Western and Eastern Cape, South Africa. Methods Phylogeographic analysis using parsimony, maximum likelihood, minimum evolution and Bayesian inferences was employed for the 16S rRNA mtDNA gene region, while bootstrapping and posterior probabilities were used to assess the robustness of clades. In addition, nested clade analysis was performed in an attempt to disentangle the contemporary and historical factors that have sculpted genealogical relationships among conspecific populations of P. perlatus. Results Phylogenetic topologies were congruent irrespective of the evolutionary method employed. Two highly distinct reciprocally monophyletic clades characterized by marked levels of corrected sequence divergence were present, with no shared haplotypes between the two major phylogroups. Phylogroup one comprises the populations of the westward-flowing drainages (mainly the Berg and Olifants drainages), and phylogroup two comprises all of the southward-flowing drainages and can further be divided into two subclades – one containing the Breede River populations, and the other containing the Gamtoos and Gourits drainage systems. The nested clade analysis demonstrated restricted gene flow and long-distance dispersal for a number of higher clade levels. The higher-level groups and results for the total cladogram suggest either fragmentation or isolation by distance. Main conclusions Freshwater crabs are generally highly philopatric, and dispersal, although not common, has occurred historically. The westward-flowing drainages (Berg, Olifants, Eerste, Liesbeeck and Tokai) are isolated from the southward-flowing drainages by the Cape Fold Mountains, while the southward-flowing drainages have a number of tributaries that extend into the low-lying regions, allowing for gene flow between these three major drainages systems (Breede, Gamtoos and Gourits). Among the westward-flowing drainages, a more intensive sampling regime is required to understand evolutionary relationships. Our molecular results suggest that the observed patterns pre-date the formation of contemporary hydrographic patterns in the Cape. This suggests that an older Late Miocene event has severely impacted the contemporary population structure in this species, as recent Pliocene hydrographic boundaries do not correspond to the phylogeographic pattern observed. Conservation efforts for aquatic taxa should clearly be directed at the catchments, in an attempt to conserve biological diversity.